The invention relates to CD40 binding proteins, which can be used as modulators of the CD40 signaling pathway and/or the CD40-induced nuclear factor kappa B (NF-kB) activating pathway and thus useful in the treatment of CD40 related diseases (e.g., inflammatory diseases) and/or NF-kB related diseases and/or in the improvement of anti-tumor treatments. The current invention also relates to nucleic acid sequences coding for the CD40 interacting proteins (also called xe2x80x9cTTRAPxe2x80x9d (xe2x80x9cTRAF and TNF receptor associated proteinxe2x80x9d) for CD40 receptor associated protein). The invention further relates to the use of the polypeptides derived from these CD40 interacting proteins in the treatment of CD40 and/or NF-kB related diseases and or cancer. Furthermore, the invention concerns pharmaceutical preparations comprising the CD40 interacting proteins or polypeptides derived from these proteins.
CD40 is a receptor of tumor necrosis factor (xe2x80x9cTNFxe2x80x9d) receptor superfamily (Banchereau et al., 1994), which is expressed at the surface of B-cells, antigen presenting cells (APC), and several non-hematopoietic cells such as endothelial cells (Hollenbaugh et al., 1995), epithelial cells (Galy and Spits, 1992), fibroblasts (Fries et al., 1995) and keratinocytes (Gaspari et al., 1996). The ligand for CD40 (CD40L) occurs mainly on activated T-cells. Up to now the role of CD40 was mainly studied in the context of the T-cell APC/B-cell interaction (for a review, see Noelle, 1996). Amongst others, the CD40-CD40L interaction seems to be important for the T-cell mediated immunity and for primary and secondary humoral immune response. These findings were confirmed by experiments in mouse models showing that treatment with anti-CD40L antibodies resulted in blocking of the development of mouse equivalents of human autoimmune diseases such as arthritis (Durie et al., 1993), oophoritis (Griggs et al., 1996) and multiple sclerosis (Gerritse et al., 1996). Activation and transduction through the CD40 pathway within this biological system is mainly responsible for B cell activation and the humoral immune response accordingly.
Apart from NF-kB, factors that can be activated by stimulation of CD40 are NF-AT (Francis et al, 1995) c-Jun, ATF-2 and IRF-1 (Karmann et al., 1996). All these factors play an important role in inflammation.
The CD40L induced signal transduction is, like TNF, mediated by the binding of TNF-Receptor Associated Factors (TRAF""s) to the cytoplasmic domain of the receptor. Chaudhuri et al. (1997) demonstrated that, at least in human B cell lines, CD40 and TRAF2 are constitutively associated with each other, and that this association is inhibited by CD40 mediated signals. Apart from the binding with TRAF2, the cytoplasmic domain of CD40, which consists of 62 amino acids at positions 196-257 (mature human CD40xe2x80x94numbering according to Kashiwada et al., 1998), is known to associate with TRAF3, TRAFS, TRAF6 and Janus kinase 3. TRAF6 binds to the amino-terminal cytoplasmic tail of CD40 at positions 210-225, although it can not be excluded that full association of TRAF6 with CD40 may also require the carboxy-terminal part at positions 226-249 (Ishida et al., 1996). TRAF2, TRAF3 and TRAF5 bind to the carboxy-terminal CD40 cytoplasmic domain at positions 226-249 (Ishida et al., 1996).
Stimulation of CD40 results in activation of protein kinases, the nitrogen-activated protein kinase and Janus kinase 3/signal transducer and activator of Transcription 3. Moreover, stimulation of CD40 mediates critical biological effects in B cell growth survival and differentiation.
It is known that TRAF2 and TRAF5 play a role in NF-kB activation in signaling through CD40, as well as xe2x80x9cTNF receptor type I (TNF-RI), TNF receptor type II (TNF-RII)xe2x80x9d CD30 and lymphotoxin b receptor. TRAF6 participates in NF-kB activation signaled by CD40 and IL-1 receptor. In addition to these data, International Patent Applications WO 96/16665 and WO 96/28568 disclose a TRAF like protein that binds to the cytoplasmic domain of CD40.
We show herein that at least two other proteins exist which unexpectedly interact with the cytoplasmic domain of CD40. Even more surprisingly, neither of these proteins shows significant homology, with any known CD40, interacting proteins. Further, no significant homology exists between the two proteins themselves. These proteins should therefore be considered as two new classes of CD40 interacting proteins.
The present invention thus concerns an isolated functional protein capable of interacting with the cytoplasmic domain of CD40 and/or other receptors of the TNF receptor superfamily such as CD30 or TNF receptor II, wherein the protein has no homology to TRAP-proteins.
The invention also includes an isolated functional protein either comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ ID NO: 2 or either comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ ID NO: 4 or in the alternative comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ ID NO: 6.
More specifically, the functional protein comprises an amino acid sequence with 70-100% homology to the amino acids 54-362 of SEQ ID NO: 2, even more specifically the functional protein comprises an amino acid sequences with 70-100% homology to the amino acids 274-362 of SEQ ID NO: 2 or in the alternative and/or comprising an amino acid sequence with 70-100% homology to the amino acids 2-245 of the SEQ ID NO: 6.
Furthermore, the invention also includes those proteins or peptides having 70-100% homology to at least, any of the three peptides as depicted in SEQ ID NO: 2 located between the residues numbering 115-121, 145-153 and 347-352 respectively. The amino acid sequence of residue numbering 115-121 is SLITWNI; the amino acid sequence of residue numbering 145-153 is PDVIFLQEV and the amino acid sequence of residue numbering 347-352 is FPSDHW.
xe2x80x9cHomologyxe2x80x9d, in this context, means identical or similar to the referenced sequence while obvious replacements/modifications of any of the amino acids provided, are included as well. A homology search in this respect can be performed with the BLAST-P (Basic Local Alignment Search Tool), a program well known to those of skill in the art. For the corresponding nucleic acid sequence, homology is referred to the BLASTX and BLASTN programs known in the art.
The invention thus relates to a DNA sequence encoding CD40 receptor associated protein or encoding an immunologically active and/or functional fragment of such a protein, selected from the group consisting of:
(a) DNA sequences comprising a nucleotide sequence encoding a protein comprising the amino acid sequence as given in SEQ ID NO: 2;
(b) DNA sequences comprising a nucleotide sequence as given in SEQ ID NO: 1;
(c) DNA sequences hybridizing with the complementary strand of a DNA sequence as defined in (a) or (b) and encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the DNA sequence of (a) or (b);
(d) DNA sequences, the nucleotide sequence of which is degenerated as a result of the genetic code to a nucleotide sequence of a DNA sequence as defined in any one of (a) to (c); and
(e) DNA sequences encoding a fragment of a protein encoded by a DNA sequence of any one of (a) to (d).
One embodiment of the invention is a protein with SEQ ID NO: 2. Another embodiment of the invention is a protein with SEQ ID NO: 4. A further embodiment of the invention concerns a protein with SEQ ID NO: 6.
A further aspect of the invention is the use of the aforementioned proteins, or biologically active fragments thereof, to modulate and/or inhibit members of the TNF receptor superfamily such as CD40, CD30 or TNF-receptor II in their signaling activity and/or CD40-induced NF-kB activation and/or JUN-kinase activity.
The isolated functional protein according to the invention and/or a functional fragment thereof can be used to treat TRAF-related, CD40-related, NF-kB related and/or Jun (kinase) related diseases. Such diseases include atherosclerosis, arthritis, multiple sclerosis, systemic lupus erythematosis (xe2x80x9cSLExe2x80x9d) and/or graft rejection.
In addition, the isolated functional protein according to the invention and/or a functional fragment thereof can be used to sensitize tumor cells to anti-tumor treatments.
Another aspect of the invention is the use of aforementioned proteins or biologically active fragments thereof to screen for compounds that interfere in the interactions of the proteins or fragments with other protein components of the CD40, TRAF or NF-kB related signaling pathway.
The invention also relates to a method for identifying and obtaining an activator or inhibitor of CD40 receptor associated proteins comprising the steps of:
(a) combining a compound to be screened with a reaction mixture containing the protein of the invention and a readout system capable of interacting with the protein under suitable conditions;
(b) maintaining the reaction mixture in the presence of the compound or a sample comprising a plurality of compounds under conditions which permit interaction of the protein with the read out system;
(c) identifying or verifying a sample and compound, respectively, which leads to suppression or activation of the read out system.
As used herein, the term xe2x80x9cread out systemxe2x80x9d means a DNA sequence which upon transcription and/or expression in a cell, tissue or organism provides for a scorable and/or selectable phenotype. Such read out systems are well known to those skilled in the art and comprise, for example, recombinant DNA molecules and marker genes as previously described herein.
The term xe2x80x9cplurality of compoundsxe2x80x9d in a method of the invention is to be understood as a plurality of substances which may or may not be identical.
The compound or plurality of compounds may be comprised in, for example, samples, e.g., cell extracts from animals or microorganisms. Furthermore, the compound(s) may be known in the art but hitherto not known to be capable of suppressing or activating CD-40 receptor associated interacting proteins. The reaction mixture may be a cell free extract or may comprise a cell or tissue culture. Suitable set ups for the method of the invention are known to the person skilled in the art and are, for example, generally described in Alberts et al., Molecular Biology of the Cell, (3rd ed. 1994). 1The plurality of compounds may be, e.g., added to the reaction mixture, culture medium or injected into the cell.
If a sample containing or a plurality of compounds, is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound capable of suppressing or activating CD40 receptor associated proteins, or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s). Preferably, the sample comprises substances of similar chemical and/or physical properties, and most preferably the substances are identical. The compounds which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra).
Another aspect of the invention involves DNA molecules, also called nucleic acid sequences, encoding for the aforementioned proteins, preferably a nucleic acid sequence with 70-100% homology to the DNA sequence depicted in SEQ ID NO: 1 and/or a nucleic acid sequence with,70-100% homology to the DNA sequence depicted in SEQ ID NO: 3 or in the alternative a nucleic acid sequence with 7-100% homology to the DNA sequence depicted in SEQ ID NO: 5.
xe2x80x9cHomologyxe2x80x9d, in this context, means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of the nucleic acid molecules are, for example, variations of the nucleic acid molecules which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally counting allelic variants as well as synthetically genetically engineered variants.
The proteins encoded by the various derivatives and variants of the above-described nucleic acid molecules have similar common characteristics, such as biological activity, molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as electrophoretic mobility, chromatographic behavior, sedimentation coefficients, pH optimum, temperature optimum, stability, solubility, spectroscopic properties, etc.
A further embodiment of the invention concerns a method for identifying and obtaining CD40 receptor associated proteins comprising the LexA two-hybrid system wherein LeXA DNA-binding domain as a bait and a HeLa cell fusion library in plasmid pJG45 as prey is used. A DNA sequence encoding a CD40 receptor associated protein obtainable by the method belong to the invention as well.
The present invention also relates to vectors, particularly plasmids,cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a nucleic acid molecule according to the invention. Methods which are well known to those skilled in the all can be used to construct various plasmids and vectors; see, for example, the techniqques described in Sambrook. Molecular Coning A Lboratory Manual Cold Spring Harbor Laboratory (1989) N.Y.
Alternatively, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
In a preferred embodiment, the nucleic acid molecule present in the vector is operably linked to a control sequence or control sequences that allow for the expression of the nucleic acid molecule in prokaryotic and/or eukaryotic cells.
The term xe2x80x9ccontrol sequencexe2x80x9d refers to regulatory DNA sequences which are necessary to affect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In procaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eucaryotes generally, control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term xe2x80x9ccontrol sequencexe2x80x9d is intended to include, at minimum all components the presence of which are necessary for expression, and may also include additional advantageous components.
The term xe2x80x9coperably linkedxe2x80x9d refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence xe2x80x9coperably linkedxe2x80x9d to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. If the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is used.
Thus, the vector of the invention is preferably an expression vector. An xe2x80x9cexpression vectorxe2x80x9d is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art. The present invention furthermore relates to host cells comprising a vector as described above or a nucleic acid molecule according to the invention wherein the nucleic acid molecule is foreign to the host cell.
By xe2x80x9cforeignxe2x80x9d it is meant that the nucleic acid molecule is either heterologous with respect to the host cell, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host cell but located in a different genomic environment than the naturally occurring counterpart of the nucleic acid molecule. This means that, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of the host cell, in particular it is surrounded by different genes. In this case the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter. The vector or nucleic acid molecule according to the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extra-chromasomally. In this respect, it is also to be understood that the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination (Paszkowski (ed.), Homologous Recombination and Gene Silencing in Plants, Kluwer Academic Publishers (1994)).
The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. 
The invention also includes a method for preparing CD40 receptor associated proteins which method comprises the cultivation of host cells according to the invention which, due to the presence of a vector or a nucleic acid molecule according to the invention, are able to express such a protein, under conditions which allow expression of the protein and recovering of the so-produced protein from the culture.
The present invention furthermore relates to proteins encoded by the nucleic acid molecules according to the invention or produced or obtained by the herein described methods, and to functional and/or immunologically active fragments of such CD40 receptor associated proteins. The proteins and polypeptides of the present invention are not necessarily translated from a designated nucleic acid sequence; the polypeptides may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system, or isolation from a suitable viral system. The polypeptides may include one or more analogs of amino acids, phosphorylated amino acids or unnatural amino acids. Methods of inserting analogs of amino acids into sequence are known in the art. The polypeptides may also include one or more labels, which are known to those skilled in the art. In this context, it is also understood that the proteins according to the invention may be further modified by conventional methods known in the art. By providing the proteins according to the present invention it is also possible to determine fragments which retain biological activity, namely the mature, processed form. This allows the construction of chimeric proteins and peptides comprising an amino sequence derived from the protein of the invention, which is crucial for its binding activity. The other functional amino acid sequences may be either physically linked by, e.g., chemical means to the proteins of the invention or may be fused by recombinant DNA techniques well known in the art.
Furthermore, the present invention relates to antibodies specifically recognizing a CD40 receptor associated protein according to the invention or parts, i e., specific fragments or epitopes, of such a protein. The antibodies of the invention can be used to identify and isolate other CD40 receptor associated proteins and genes in any organism. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kxc3x6hler and Milstein, Nature 256 (1975), 495, and Galfxc3xa9, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d, CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the imnmunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention. For example, surface plasmon resonance as employed in the BLAcore system can be used to increase the efficiency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995). 7-13). In many cases, the binding phenomena of antibodies to antigens is equivalent to other ligand/anti-ligand binding.
The invention also relates to a diagnostic composition comprising at least one of the aforementioned nucleic acid molecules, vectors, proteins, antibodies or compounds and optionally suitable means for detection.
The diagnostic compositions may be used for methods for detecting expression of related CD40 receptor associated proteins by detecting the presence of the corresponding mRNA which comprises isolation of mRNA from a cell and contacting the mRNA so obtained with a probe comprising a nucleic acid probe as described above under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the protein in the cell. Further methods of detecting the presence of a protein according to the present invention comprises immnuno-techniques well known in the art, for example enzyme linked immunosorbent assay.
The invention also relates to a pharmaceutical composition comprising one or more of the above mentioned proteins or fragments in a biologically active amount for the treatment of CD40, TRAF and/or NF-kB related diseases such as atherosclerosis, arthritis, multiple sclerosis, systemic lupus erythematosis, graft rejection and the like.
In another aspect, the present invention relates to a pharmaceutical composition comprising one or more compounds obtainable by the above mentioned screening method for the treatment of CD40, TRAF and/or NF-kB related diseases such as atherosclerosis, arthritis, multiple sclerosis, systemic lupus erythematosis, graft rejection and the like.